Nutrient composition and pattern of food intake may play a significant role in weight gain. The aim of this study was to document the effects of a daily 3-meal pattern with isocaloric diets containing different dietary protein contents on growth performance and different plasma biochemical indexes including amino acid plasma concentration in castrated male pigs. Then, 21 DLY (Duroc×Landrace×Yorkshire) pigs aged 60 days were assigned randomly into 3 groups: a control group , a group receiving high then basal and then low CP meals (High-Low group) and a group receiving low then basal and then high CP meal (Low-High group) for 40 days with pigs being feed-restricted. On day 40, after 12 h fasting, blood samples were obtained for analysis. The results showed that the insulin/glucagon ratio was lower in the High-Low group (P<0.05) when compared with the control group. Compared with the control group, the average daily gain of pigs from the High-Low group increased by 14.10% (P = 0.046). Compared with the control group, serum gamma-glutamyl transferase (GGT) decreased significantly (P<0.05) in both the High-Low and Low-High groups. Plasma concentrations of branched-chain amino acids  increased in the Low-High group (P<0.05) when compared with the control group; and plasma methionine and serine decreased in both the two experimental groups (P<0.05). Compared with the High-Low group, all the BCAA increased significantly (P<0.05) in the Low-High group. These findings suggest that the sequence and quantity of alimentary protein intake affect the insulin/glucagon ratio, as well as amino acid concentrations including BCAA, methionine and serine. It is proposed that meal pattern with pigs receiving high then basal and then low CP meals daily may help to improve the weight gain of pigs. Pattern of nutrient consumption may reset peripheral circadian clocks and the related metabolic processes . DietaryAll aspects of physiology, including sleep-wake cycles, cardiovascular activity, endocrine system, body temperature, renal activity, gastrointestinal tract motility, and metabolism, are influenced by the circadian clock . Genome-This represents a paradigm shift in pig feeding, since the optimal dietary nutrient level is more and more considered as a dynamic process. Hence, we made the working hypothesis that a daily 3-meal pattern with different dietary protein contents may affect biochemical circulating parameters as well as body physiology and metabolism. Blood metabolite concentrations may help in interpretating some metabolic changes associated with different dietary modifications. Thus, the present study reports on the effects of feeding a daily 3-meal pattern with different dietary protein contents daily on several parameters including blood insulin, glucagon, and AA concentrations in the growing castrated male pigs.Twenty-one DLY (Duroc×Landrace×Yorkshire) barrows aged 60 days were obtained from Hunan New Wellful Co., Ltd.  . The pigThe nutrients were adequate for pigs and met the NRC-recommended requirement within the weight range used in the present study . The control diet was balanced on the calculated content of digestible energy (DE) (13.72 MJ/kg), CP (181.1 g/kg), apparent ileal digestible limiting amino acids , Ca and P . The conAll animals had free access to drinking water. The dietary intervention lasted 40 d.g for 10 min at 4°C and then stored immediately at –80°C until assayed [Body weights of individual pigs were measured immediately before feeding at the beginning and end of the trial. On day  assayed ,17.Feed intake and average daily gain (ADG) were recorded, and feed intake/ADG (F/G) was calculated for all pigs.No animals were sacrificed in this study. This study was performed in accordance with the Chinese guidelines for animal welfare and approved by the Animal Care and Use Committee of the Institute of Subtropical Agriculture, the Chinese Academy of Sciences.An Automated Biochemistry Analyzer  was used to determine the concentrations of serum glucose, urea nitrogen, gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), aspartate aminotransferase (AST) activities, total protein according to the commercial kits and manufacturer’s instructions . All theSerum insulin and glucagon were determined by radioimmunoassay according to corresponding reagent kit manufacturer’s instructions  .4, followed by addition of 0.25 ml of 2 M K2CO3[o-phthaldialdehyde and fluorescence detection[Plasma (0.5 mL) was deproteinized with 0.5 mL of 1.5 mM HClO2 M K2CO3. The neudetection. Amino aData are presented as the mean ± SEM obtained from triplicate experiment. Differences between mean values of multiple groups were analyzed by one-way analysis of variance (ANOVA) followed by Post-hoc Tukey tests for multiple comparisons when interactions were significant using SPSS 13.0. DiffereThe effects of pattern of consumption of diets with different protein contents daily on body weigh are shown in Compared with the control and Low-High groups, F/G in the high-low group was not significantly different but tended to modestly decrease (0.05 < P < 0.1) by 12.3% and 10.5%, respectively .Serum biochemical indices are shown in Compared with the Low-High group, serum LDH in the High-Low group was lower (P < 0.05). Compared with the control group, serum urea concentration was not significantly different in the High-Low group and Low-High group but tended to increase by 20.14% and 16.67% .Serum hormone concentrations are shown in For the essential amino acids, compared with the control group, plasma valine was higher in the Low-High group (P < 0.05), plasma leucine was lower in the High-Low group (P < 0.05) and plasma methionine was lower by 20.4% (P < 0.05) and 19.4% (P < 0.05) respectively in the High-Low and Low-High .For the non-essential amino acids, plasma serine and taurine were lower in the two experimental groups (P < 0.05) . DiffereSimilarly, according to the dietary pattern, diets containing an equivalent amount of nutrients had different effects on several indicators of the metabolism of amino acids . Indeed,to the control group and, since caloric intake was an equivalent food efficiency also tended to be increased. In a recent study, modifications of the daily-phase feeding system did not significantly modify body weight but reduced N excretion by 12% (P < 0.01) [Circadian clock plays a crucial role in the regulation of numerous physiological processes and the beneficial versus deleterious effects of a given diet may be partly related to the sequence of dietary intake in the daytime . Circadi < 0.01) . Whateve < 0.01) . High-CP < 0.01)  and huma < 0.01) . In the Transamination in cells and tissues is accomplished by enzymes called transaminases or aminotransferases. This process is an important step in the synthesis of some non-essential amino acids and urea synthesis. The activities of enzymes as well as some endocrine responses are related to these metabolic pathways and are also known to be modified by time-restricted feeding . ElevateIt is noteworthy that behaviors and physiological activities in animals display circadian rhythms, allowing the organisms to anticipate and prepare for the diurnal changes in the living environment . Plasma It is well known that proteins have a notable impact on glucose homeostasis mechanisms, predominantly through their effects on insulin, incretins, gluconeogenesis, and gastric emptying . InsulinPrevious studies have shown that the glucagon response to feeding with protein depends on the increase in plasma amino acid concentrations . It has The findings reported in the present study strongly suggest that the varying the protein content in the meal affects the insulin/glucagon ratio, and amino acids including BCAA, methionine and serine in pigs. In this study, it was found that the varying meal protein content affects circulating biochemical parameters, which may impact the growth performance of pigs, and then may be useful for pig production. In practice, this study suggests that the High-Low feeding procedure improved growth and feed efficiency.


MicroRNAs (miRNAs) are short, highly conserved small noncoding RNAs that had fundamental roles in post-transcriptional gene expression, and they are crucial for proper control of biological processes and known to participate in embryo implantation. However, miRNA expression profiles in the pre-receptive and receptive phases of the goat endometrium during embryo implantation are unknown.P-values< 0.05. Moreover, GO and KEGG analysis of the target genes of the differentially expressed miRNAs revealed some candidate miRNAs, genes and pathways that may involve in the formation of the receptive endometrium. Based on stem-loop RT-qPCR, 15 miRNAs were detected and the results suggested that the majority of the miRNA expression data measured by Solexa deep sequencing could represent actual miRNA expression levels.A total of 1,069 and 847 miRNAs were expressed in receptive (R) and pre-receptive (P) goat endometrium, and 632 miRNAs were co-expressed in both phases. We identified 545 (50.98%) known miRNAs in the R library and 522 (61.63%) in the P library. There were 110 up-expressed miRNAs and 33 down-expressed miRNAs in receptive endometrium compared with the pre-receptive endometrium meeting the criteria of Our data revealed the first miRNA profile related to the biology of the goat receptive endometrium during embryo implantation, and the results suggested that a subset of miRNAs might play important roles in the formation of endometrial receptivity. Thus, elucidating the physiological roles of endometrial miRNAs will help us better understand the genetic control of embryo implantation in goats. Embryo implantation is a crucial step in mammalian reproduction . EndometSome factors act directly on the endometrium, and regulated endometrial functions promote the interactions between endometrium and embryo . AccumulEndometrial receptivity is a dynamic process that was believed to be under post-transcriptional regulation because several genes expressed in the endometrium were identified as being epigenetically regulated . MicroRNNevertheless, little information was available on miRNA expressed in the goat endometrium, and differences between the pre-receptive and receptive phases during embryo implantation were unknown. Focusing on these points, we aimed to gain more understanding of the complex regulation of endometrial receptivity in healthy multiparous dairy goats by analyzing miRNA expression in the endometrium at gestational day 5 (pre-receptive endometrium phase) and gestational day 15 (receptive endometrium phase) using Illumina Solexa technology. And then we investigated the known miRNAs, potential novel miRNAs, differentially expressed miRNAs and their target genes. Given the biological functions of miRNAs at the posttranscriptional level and genes usually interact with each other to play their roles, GO enrichment and KEGG pathway analysis were also made in this study. The results would provide further understanding of the miRNAs role in the regulation mechanism of the endometrial receptivity.ftp://mirbase.org/pub/mirbase/CURRENT/), the Rfam database (http://rfam.janelia.org), the Repbase database (http://www.girinst.org/repbase), the Genome database (http://goat.kiz.ac.cn/GGD/download.htm) and the mRNA database (http://goat.kiz.ac.cn/GGD/download.htm). Small RNAs were classified into different categories according to their annotations. Next, 3’ ADT and length filter, junk reads, Rfam, mRNA, repeats, rRNA, tRNA, snRNA and snoRNA sequences, as well as other Rfam RNA sequences, were separated out and discarded. As a result, 4,233,874 clean reads representing 284,622 unique sequences from the R library were obtained, and 5,386,427 clean reads representing 139,040 unique sequences from the P library  and P (pre-receptive) endometrium, respectively. To assess the efficiency of Solexa sequencing and the quality of the sequences, all reads were annotated and classified by aligning against the miRBase 20.0 database  Group 1b, containing 14 miRNAs corresponding to 8 pre-miRNAs and miRNAs in miRBase 20.0; the pre-miRNAs further map to the genome and EST. (2) Group 2a, containing 215 miRNAs corresponding to 174 pre-miRNAs and miRNAs in miRBase 20.0; the mapped pre-miRNAs do not map to the genome, but the reads (including the miRNAs of the pre-miRNAs) map to the genome. The extended genome sequences from the genome loci may form hairpins. (3) Group 3a, containing 326 miRNAs corresponding to 270 pre-miRNAs and miRNAs in miRBase 20.0. The mapped pre-miRNAs and the reads do not map to the genome. (4) Group 4a, containing 621 miRNAs corresponding to 636 pre-miRNAs. Reads do not map to selected pre-miRNAs in miRbase 20.0, but do map to the genome. The extended genome sequences might form hairpins . From thftp://mirbase.org/pub/mirbase/CURRENT/). Sequences with a perfect match or one mismatch were retained in the alignment. We identified 545 known miRNAs in the R library and 522 in the P library  in miRBase 20.0 ( library . Conside library .The expression levels of oar-miR-10a_R+1_1ss12TA (a variant of oar-miR-10a as described previously in the same way ), oar-mihttp://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi/). A total of 643 miRNA candidates were identified as having the typical stem-loop secondary structure, with 488 candidates identified in the R library and 298 in the P library |> 1, and a total of 143 differentially expressed miRNAs were selected from the P and R libraries  increase in the receptive endometrium compared with the pre-receptive endometrium. Other miRNA that were differentially expressed between our two libraries included bta-miR-183_L-1, PC-5p-20799_45, hsa-miR-184, PC-3p-25064_36, hsa-miR-187-3p_R+1, oan-miR-429-3p_R+2, PC-5p-5933_158, hsa-miR-200a-5p, PC-3p-36269_24, oar-miR-154b-3p_L-1 and so on . MoreoveFor animal miRNAs, sequence complementarities with their targets were usually restricted to the 5’ region, particularly those at nucleotide positions 2 through 7, and it was likely that many miRNAs function through cooperative regulation of multiple mRNAs . Owing tWe used a well-established miRNA-target dataset to investigate the possible function of these miRNAs with the aid of gene function annotation methods, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) progress.GO enrichment analysis using cellular components showed that 490 genes (13.64%) mapped to GO terms for component topology in the database. The analysis of biological processes showed that 2,087 genes (58.10%) were involved in cellular or metabolic processes, and the analysis of molecular function showed that 1,015 genes (28.26%) were assigned different functions based on gene background . GO biolKEGG pathway annotation showed 1,886 target genes that were annotated for 220 biological processes . MoreoveIn general, qRT-PCR is considered the standard method to detect gene expression. To validate the reliability of the sequencing data, we applied Stem-loop RT-qPCR to compare the expression levels of the differentially expressed miRNAs and newly identified miRNAs. Five conserved miRNAs  and ten potential novel miRNAs  were selected due to these miRNAs differential expression between R and P libraries, and may participate in the formation of endometrial receptivity. The expression levels of twelve miRNAs  in the receptive endometrium determined by Solexa deep sequencing or qRT-PCR were higher than those in pre-receptive endometrium . HoweverTo date, several miRNAs have been identified in the goat , 30, butAfter separating out and discarding junk sequences, we obtained 4,233,874 clean reads representing 284,622 unique sequences from the R library and 5,386,427 clean reads representing 139,040 unique sequences from the P library . The lenftp://mirbase.org/pub/mirbase/CURRENT/). A total of 1,284 miRNAs were identified: 632 of these were co-expressed in both libraries, 1,069 miRNAs were detected in the R library and 847 in the P library. Compared with previous research, more conserved miRNAs  were identified in this study [Next, we aligned the clean reads to the miRNAs of all known animals in the miRBase 20.0 database (is study . There aP< 0.05), and there were 110 miRNAs increased at least a 2-fold in expression. Xia reported that 28 miRNAs were up-regulated at least two-fold and 29 miRNAs were down-regulated at least two-fold during the receptive phase compared with the pre-receptive phase in the rat uterus [To investigate differences in endometrium activity in the receptive and pre-receptive phases, differentially expressed miRNA were identified in the two constructed libraries. In the present study, 292 miRNAs were up-regulated and 150 miRNAs were down-regulated in the receptive phase compared with the pre-receptive phase  and increased 14.55-fold compare with pre-receptive endometrium. Studies have identified that miR-182 was significantly up-regulated in endometrial carcinoma tissues (EC) compared with complex atypical hyperplasia, simple hyperplasia and normal endometrial tissues –50. Furt8.63 and Next, we turned our attention to the specific expressed miRNAs. The miRNA with the highest tissue-specific expression was bta-miR-431_R-3 from the R library. After careful analysis, we found that its sequence was consistent with miR-431, which was initially identified as a central nervous system specific miRNA cloned from the brain tissue of mouse embryos . Wu repoThe highly tissue-specific miRNA in the P library was bta-miR-216a_R-2, whose sequence was identical to miR-216a with one mismatch. This miRNA regulates Ybx1 post-transcriptionally, which is a key mechanism for enhanced mRNA translation in kidney cells . AnotherOne of the most important characteristics that distinguishes miRNAs from other endogenous small RNAs is the capacity of the precursor miRNA sequence to fold back into a canonical stem-loop hairpin structure . In thisPrevious studies showed that miRNAs modulated gene expression by inhibiting mRNA translation or regulating mRNA degradation at the posttranscriptional level based on Watson-Crick pairing between the 5’ end of the miRNA  and the 3’ un-translated regions (3’ UTR) of target mRNAs . An animGO is an international standardized classification system for gene function, supplying a set of controlled vocabulary to comprehensively describe the properties of genes and gene products . GO analIn organisms, genes usually interact with each other to play different roles in certain biological functions . KEGG paWe performed stem-loop qRT-PCR, which is a reliable method used to detect, measure and validate the expression levels of miRNAs. The expression trends of 8 miRNAs measured by Solexa deep sequencing were consistent with those determined by stem-loop qRT-PCR, but the differences in expression levels between the two methods did not agree well. Moreover, the expression patterns of miR-200a measured by Solexa deep sequencing were opposed to those determined by stem-loop RT-qPCR.This inconsistency might result from differences in the two methods. The expression levels of miRNAs measured by Solexa deep sequencing required the following seven steps: RNA extraction, small RNA fractionation, 5’ adaptor ligation, 3’ adaptor ligation, RT-PCR, sequencing, and data analysis; thus, the information from some sequences might be lost or produce low-quality tags that were filtered during data analysis . MoreoveWe obtained high-quality miRNA expression profiles from pre-receptive endometrium and receptive endometrium, and predicted 545 known and 33 novel miRNAs in endometrium of dairy goats for the first time. Target gene predictions for the 143 differentially expressed miRNAs, functional annotations and pathway analyses in GO and KEGG databases could contribute to a better understanding of the miRNA mediated regulation of target genes in the development of the endometrium receptivity.All animals in this study were maintained according to the No. 5 proclamation of the Ministry of Agriculture, P. R. China. And animal protocols were approved by the Review Committee for the Use of Animal Subjects of Northwest A&F University.A total of 20 healthy 24-month-old multiparous dairy goats (Xinong Saanen) were induced to estrous synchronization for this study. The first day of mating was considered to be Day 0 of pregnancy. Gestational days 5 and 15 were important time points for the embryo implantation in goats . The expTotal RNA was extracted from every sample using Trizol reagent  following the manufacturer’s procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100  and RNA 6000 Nano LabChip Kit  with RIN number> 7.0. Approximately 1 ug of total RNA were used to prepare small RNA library according to protocol of TruSeq Small RNA Sample Prep Kits . The total RNA with lowest quality was not used for further study from the pre-receptive endometrium samples and receptive endometrium samples, respectively. P library was constructed by mixing the nine samples into three samples with the same concentration, performing the single-end sequencing (36 bp) on an Illumina Hiseq2500  thrice at the LC-BIO  following the vendor’s recommended protocol, and R library was constructed in the same way.LC Sciences Service. Briefly, the raw reads were subjected to the Illumina pipeline filter (Solexa 0.3), and then the dataset was further processed with an in-house program  to remove adapter dimers, junk, low complexity, common RNA families  and repeats.Data processing followed the procedures described in a previous study  by LC Scftp://mirbase.org/pub/mirbase/CURRENT/) by BLAST search to identify known miRNAs and novel 3p’ and 5p’ derived miRNAs. The remaining sequences were mapped to other selected species precursors (with the exclusion of specific species) in miRBase 20.0 by BLAST search, and the mapped pre-miRNAs were further BLASTed against the specific species genomes to determine their genomic locations. The above two were defined as known miRNAs. On the basis of analysis for detected miRNAs, we further analyzed the conservatism of these miRNAs with selected species on frequency for the number in statistics.Unique sequences 18–26 nt in length were mapped to specific species precursors in miRBase 20.0 (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi/). The criteria for secondary structure prediction were described by Mi and Lian [Sequencing reads that did not match any known miRNAs were further analyzed to discover novel miRNAs using the characteristic hairpin structure of the miRNA precursor , 58. To and Lian , 70.To compare differentially expressed miRNAs in the receptive endometrium (R library) and pre-receptive endometrium (P library) of goats, the expression abundances of miRNAs in the two samples were normalized to obtain the expression of transcripts per 1,000,000. And the formulae was: Normalized expression (NE) =  × 1,000,000.P-value for each miRNA were calculated based on the normalized expression using the formulae as described previously [When the NE of a certain miRNA was 0 in one of the two libraries, we revised the 0 to 0.001 for the comparative, and the miRNA whose NE was less than 1 in both libraries were discarded described in detail previously . The difeviously  and showP-value formula:N1 and X represent the total number of clean reads and normalized expression level of a given miRNA in the small RNA library generated from P library, respectively, and N2 and Y represent the total number of clean reads and normalized expression level of a given miRNA in the small RNA library generated from the R library, respectively.chttp://www.targetscan.org/cgi-bin/targetscan/data.download.cgi?db=vert.61 and miRanda 3.3a chttp://www.microrna.org/microrna/home. do) were used to predict target genes of differentially expressed miRNAs. Only when the target was identified by both programs, it was considered to be the predicted target for a given miRNA.Two computational target prediction algorithms (TargetScan 5.0 ftp://ftp.ncbi.nih.gov/gene/DATA/gene2go.gz), calculated gene numbers for each term and used hyper-geometric tests to find significantly enriched GO terms in target gene candidates. The results were compared with the reference gene background and to genes corresponding to certain biological functions.GO enrichment analysis was performed on predicted target gene candidates of differentially expressed miRNA. This method mapped all target gene candidates to GO terms in the database  used the same formula as GO analysis to determine target gene candidates. Here, N was the number of all genes with KEGG annotation, n was the number of target gene candidates in N, M was the number of all genes annotated in a certain pathway and m was the number of target gene candidates in M [KEGG pathway analysis facilitates the understanding of biological functions of genes . The anates in M .http://www.cytoscape.org/) to analyze the interactions between miRNA and genes. Firstly, the relationship and the interactions between miRNAs and genes were determined by using their differential expression values in the databases of TargetScan 5.0 and miRanda 3.3a. And then we used the random variance model (RVM) corrected t-test (P< 0.05) to identify the differently expressed genes and miRNAs from the gene expression profiles. The overlap between the genes whose expression was induced and the target genes whose expression was reduced by miRNA were then chosen to construct the network based on the two respective expression values by using the miRNA–mRNA modules via population-based probabilistic learning methods [The miRNA-gene network was constructed using Cytoscape Software  , 1μl PrimeScript RT Enzyme Mix 1, 1μl Stem-loop primer and 4 μl RNase-Free dH2O to a final volume of 20 ul, incubated the mixture at 42°C for 15 min, followed by 85°C for 5 sec. The cDNA products were diluted 1:10 (v/v) with sterile water.Stem-loop qPCR was used to validate the known and potential novel miRNAs in present manuscript. Total RNA was extracted using Trizol reagent  following the manufacturer’s instructions. Total RNA was converted to cDNA with a Stem-loop primer using the Prime Script RT reagent Kit with gDNA Eraser  according to the manufacturer's instructions. The reverse transcription reaction system were: added 5 μl total RNA (800 ng), 2 μl 5× gDNA Eraser Buffer, 1μl gDNA Eraser and RNase-Free dH2O. The reaction mixtures were amplified in a 96-well plate at 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, 60°C for 1 min, and all reactions were performed in triplicate. 18S rRNA was used as the reference, and all primers for the Stem-loop RT-qPCR are shown in -ΔΔCt, ΔΔ = (CT miRNA − CT 18s) R − (CT miRNA − CT 18s) P. The Ct (cycle threshold) was defined as the cycle number at which the fluorescence intensity passed a predetermined threshold [RT-qPCR was performed using the Bio-Rad CFX 96 Real Time Detection System and SYBR Green PCR Master Mix  in a 20 μl reaction according to the manufacturer’s instruction. The PCR mixture included 4 ul cDNA for each miRNA, 2 μl primer mix (10 uM), 10 μl 2× SYBR Green Mix with ROX, and 4 μl RNase-Free dHhreshold .P was <0.05 and very significant when P was <0.01.All the data were processed with SPSS 17.0 . One-way ANOVA was used to compare the differences, and the method of the least significant difference (LSD) was used for further analysis, and the differences were considered significant when S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S6 Table(XLS)Click here for additional data file.S7 Table(XLS)Click here for additional data file.


Saccharomyces boulardii (S. boulardii) has been prescribed for the prophylaxis and treatment of several infectious diarrheal diseases. Gastrointestinal anthrax causes fatal systemic disease. In the present study, we investigated the protective effects conferred by Saccharomyces boulardii CNCM I-745 strain on polarized T84 columnar epithelial cells intoxicated by the lethal toxin (LT) of Bacillus anthracis. Exposure of polarized T84 cells to LT affected cell monolayer integrity, modified the morphology of tight junctions and induced the formation of actin stress fibers. Overnight treatment of cells with S. boulardii before incubation with LT maintained the integrity of the monolayers, prevented morphological modification of tight junctions, restricted the effects of LT on actin remodeling and delayed LT-induced MEK-2 cleavage. Mechanistically, we demonstrated that in the presence of S. boulardii, the medium is depleted of both LF and PA sub-units of LT and the appearance of a cleaved form of PA. Our study highlights the potential of the S. boulardii CNCM I-745 strain as a prophylactic agent against the gastrointestinal form of anthrax.The probiotic yeast  Bacillus anthracis, the causative agent of anthrax, is a large, Gram-positive, rod-shaped, aerobic, spore-forming bacterial pathogen. B. anthracis can infect a large variety of hosts by cutaneous, gastrointestinal, and pulmonary  routes. The infection progresses into a fatal systemic disease [B. anthracis-contaminated meal. There are two forms of gastrointestinal anthrax: oropharyngeal and intestinal. In the intestinal form, anthrax spore deposits can cause ulcerative lesions anywhere from the jejunum to the cecum [ disease ,2,3. Thehe cecum . In the he cecum . Early dB. anthracis synthesizes a three-component toxin as major virulence factor [N-terminus of most mitogen-activated protein kinase kinases (MAPKK or MEK) and NLRP1B, a key component of the inflammasome system [83 binds to a specific cell receptor  and ANTXR2 or CMG2 (capillary morphogenesis gene-2)), allowing its cleavage by an extracellular furin protease to a 63-kDa form (PA63) [e factor ,6,7. Hepe factor . LF is ae system ,10. Inhie system ,12,13,14Saccharomyces boulardii (S. boulardii) has been prescribed for the past 30 years for prophylaxis and treatment of diarrheal diseases caused by bacteria. Clinical evidence of the efficacy of this probiotic yeast was recently reviewed by McFarland [S. boulardii has an arsenal of anti-pathogenic strategies that could be classified into three principal areas: luminal action, trophic action and mucosal anti-inflammatory action. In a toxin-mediated disease, S. boulardii may act in the intestinal lumen by blocking the receptor or by the direct destruction of the pathogenic toxin. S. boulardii has been shown to synthesize several proteases: a 54-kDa serine protease that directly degrades toxins A and B from C. difficile and their receptors [E. coli [S. boulardii has been shown to interfere with several signaling pathways activated by bacterial toxins and implicated in cellular responses to the infection: (i) S. boulardii decreased adenylate cyclase activation and consequently chloride secretion induced by CT [S. boulardii decreased the activation of ERK1/2 mitogen-activated protein kinase and consequently IL-8 secretion induced by C. difficile toxin A [S. boulardii reversed the drop in the intestinal permeability of the human colonic mucosa after exposure to toxins A and B from C. difficile [The nonpathogenic yeast cFarland . Experimeceptors ,17,18 an[E. coli . Toxin a[E. coli . Finallyed by CT ,22; (ii) toxin A ; and ) before LT intoxication, we observed a limited effect on the reorganization of the actin cytoskeleton  against intoxication. As previously shown, the treatment of HUVECs with LT for 24 h induced a strong remodeling of the actin cytoskeleton into stress fibers   A. NotablLT alone A. We nexLT alone B. As expcleavage A. In T84oulardii A. In botcleavage A,B. An ocleavage A,B.S. boulardii protects cells via a direct interaction with the LT subunits, PA or LF, we performed a series of co-incubation experiments. Yeast cells were incubated with PA or LF for different times before assessment of the amount of toxin co-precipitated with the yeast fraction or quantification of the toxin present in the culture supernatant . S. boulardii, we did not detect PA in the pellet. A protein that was recognized by the anti-PA antibody and that migrated to the same distance as PA was present after 2, 6 and 24 h of co-incubation with S. boulardii. To investigate whether the adhesion of PA depends on the quantity of yeast, we performed an experiment in which the same quantity of PA was added to an exponentially grown yeast culture containing 107 CFU/mL or to a stationary culture containing 109 CFU/mL. S. boulardii, at all incubation time points. Moreover, a 24-hour co-incubation of PA with S. boulardii (last line) showed a supplementary band of lower molecular weight (approximately 63 kDa), probably corresponding to the cleaved form of PA.To investigate whether S. boulardii cell fraction  effective anti-toxin compounds [S. boulardii, which is currently used in the treatment of gastro-intestinal infectious diseases.Currently available prophylactic and therapeutic options to combat anthrax are limited. The only approved treatment is protective antigen targeting with a monoclonal antibody (raxibacumab), which is mostly used to treat inhalational anthrax . Better ompounds ,27. DataB. anthracis demonstrates an intrinsic ability to compromise a variety of epithelial and endothelial barriers that are present in mammalian organ systems. It is widely viewed that the anthrax toxin released by replicating B. anthracis contributes greatly to the systemic infection by disrupting the integrity and functionality of various barrier tissues. In vivo treatment of several animal species with the LT toxin leads to mortality [et al., have extensively characterized the effects of LT on intestinal tissue [Systemic dissemination is possible for each of the distinct forms of a clinical anthrax infection. In this regard, ortality ,28. Usinl tissue . This grS. boulardii preserves the barrier function of the T84 cell monolayers during LT intoxication and maintains the ZO-1 perijunctional distribution.In endothelial and epithelial cells, LT alters the cellular barrier function by affecting the tight junction (TJ) and adherens junction (AJ) complexes that consist of transmembrane proteins linking adjacent cells to the actin cytoskeleton ,29,30,31S. boulardii. Kinetic studies of LT-induced MEK-2 cleavage showed that cleavage occurred rapidly in the HUVECs at 2 h after incubation. MEK-2 cleavage was not observed in the T84 cells in this time frame. Rather, the cleavage of MEK-2 in these cells occurred after 6 h of exposure to the toxin. When both cell lines were incubated concomitantly with S. boulardii and LT, we did not observe any effect of the yeast on LT-induced MEK-2 cleavage. However, when both cell lines were exposed to the yeast before incubation with the LT toxin, MEK-2 cleavage was reduced in cells incubated with the yeast. Hence, the protective effect of S. boulardii against LT infection requires pre-incubation with the yeast. This observation is not surprising and can be explained by the release of proteases into the medium by the replicating yeast. This hypothesis is supported by the presence of active peptide in the yeast supernatant in metabolically active S. boulardii grown for at least 24–48 h [The effect of LT on the actin cytoskeleton is a relatively recent discovery. Most of the studies focused on endothelial and epithelial cells and on immune cells such as macrophages or neutrophils. After exposure to LT, human endothelium and lung epithelial cells demonstrate progressive mechanical stiffness, blebbing and endothelial barrier disruption ,25,29. T 24–48 h .S. boulardii acts on PA in two ways: (i) by adhesion of PA to the cell wall and (ii) by inducing its cleavage. Because PA pre-pore formation is necessary for LF or EF binding and translocation, the unregulated cleavage of PA and its binding to S. boulardii could dramatically interfere with toxin action. The cleavage of PA by S. boulardii requires the actions of a protease. Yeasts synthesize proteases that are implicated in protein maturation. For example, Kex-2 is a pro-hormone that processes a serine protease that is secreted by the budding yeast Saccharomyces cerevisiae. Interestingly, Kex-2 shares structural similarities with the bacterial protease subtilisin and mammalian furin [Saccharomyces boulardii CNCMI-745, Castaglioulo et al. [C. difficile toxins A and B. Thus, we hypothesize that this or another protease secreted by S. boulardii is implicated in the cleavage of PA. S. boulardii, in addition to promoting PA cleavage, can also bind to PA. This association did not depend on the growth stage of the yeast , but the quantity of PA that bound to the yeast depended on the number of yeast present. Cholera toxin (CT) is another A/B type toxin that adheres to the S. boulardii cell wall through the B subunit, leading to the internalization of the A subunit necessary for cAMP signaling and trehalase activation in yeast [S. boulardii expresses a receptor structurally and functionally similar to the enterocyte receptor for the CT—ganglioside receptor GM1. The molecule of S. boulardii that is implicated in PA adhesion remains to be determined.We show in this study that an furin . In Sacco et al.  identifiin yeast . These aS. boulardii during an anthrax infection are the binding and hydrolysis of the anthrax toxins.Taken together, our data suggest that the two fundamental protective mechanisms of The T84 human colonic cell line was obtained from the European Collection of Animal Cell Cultures  and grown in a 1:1 mixture of Dulbecco-Vogt modified Eagle’s medium and Ham’s-F12 medium (DMEM/F12) supplemented with 50 μg/mL penicillin, 50 μg/mL streptomycin  and 4% foetal bovine serum  as previously described . Human uS. boulardii were inoculated from lyophilized yeast  and grown overnight at 37 °C, with shaking in Halvorston minimal medium with 2% glucose. In the preventive protocol prior to incubation with the toxins, yeast was added to the cell culture monolayer at a ratio of 1 yeast/cell and grown overnight to a ratio of 10 yeasts/cell.Cultures of Recombinant toxins (LF and PA) were synthesized as previously described . The LT 2 porous filter membranes  (Sigma). Trans-epithelial resistance (TER) was measured using a Millicell-ERS apparatus . Under these conditions high TER values (>1000 Ω cm2) were consistently obtained in monolayers 7 days after post-seeding. The toxins and yeast were added to the apical compartment of the filter-grown cells, and TER was measured at different time as indicated in the figure.T84 cells were grown on collagen-coated 0.33 cmS. boulardii were grown as previously described, harvested by centrifugation and re-suspended in DMEM/F12 without serum. A yeast suspension (50 μL) containing 0.5 × 107 CFU/well was added to a 12-well culture plate containing 450 μL of DMEM/F12 without serum and grown overnight. The following day, LF or PA were added to the wells. Wells containing PA or LF alone and a well containing S. boulardii alone were set up as controls. After 2, 6 and 24 h of incubation, we centrifuged (1500 rpm) a 100 μL sample from each well, added running buffer to the supernatant and applied the sample to an SDS gel to detect the proteins. To determine whether the toxins could bind to the yeast, the pellets containing the yeast were washed several times in phosphate buffer, and the pellets were lysed in running buffer and applied to an SDS gel. PA detection was performed by western blotting using an anti-PA antibody , and LF detection was performed after staining the gel with Coomassie blue.3VO4, 1 mM EDTA, 1 μM aprotinin, 25 μM leupeptin, 1 μM pepstatin, 1 mM AEBSF, 10 mM NaF, 5 mM NaPPi, 10 mM β-glycerophosphate). The lysate was sonicated and solubilized for 30 min at 4 °C and then centrifuged at 14,000 rpm for 20 min at 4 °C. The protein concentration of the supernatant was determined using Bio-Rad DC reagents . Equal amounts (50 μg) of whole cell lysates were subjected to a 12% SDS-polyacrylamide gel. The proteins were transferred onto a polyvinylidene fluoride membrane  and incubated overnight at 4 °C with anti-MEK-2-N20  or anti-PA  primary antibodies followed by HRP-conjugated anti-rabbit or anti-mouse secondary antibodies, respectively . The blots were developed using the Enhanced Chemiluminescence detection system .Intoxicated cells (T84 cells or HUVECs) were washed with cold PBS and scraped with lysis buffer . The actin cytoskeleton was labelled using 1 μg/mL TRITC-conjugated phalloidin (Sigma). Immunosignals were analyzed with the LSM510-Meta confocal microscope  with a 63× magnification lens. Each picture represents the projection of six serial confocal sections.p < 0.05.All experiments were repeated at least three times. The results are presented as the mean ± SEM. Statistical significance was determined by analysis of variance with the StatView program, followed by post hoc comparison with the Bonferroni and Dunn tests. The level of significance was


The areas under the receiver operating characteristic curve of the signature were 0.86, 0.74 and 0.87 for the training, testing and the external validation stages (32 GC VS. 18 NCs), respectively. The five miRNAs were consistently dysregulated in GC tissues (n = 30). Moreover, miR-185 was decreased while miR-20a, miR-210 and miR-92b were increased in arterial plasma (n = 38). However, none of the miRNAs in the exosomes showed different expression between 10 GC patients and 10 NCs. In conclusion, we identified a five-miRNA signature in the peripheral plasma which could serve as a non-invasive biomarker in detection of GC.The differential expression of microRNAs (miRNAs) in plasma of gastric cancer (GC) patients may serve as a diagnostic biomarker. A total of 33 miRNAs were identified through the initial screening phase  pool) using quantitative reverse transcription polymerase chain reaction (qRT-PCR) based Exiqon panel (miRCURY-Ready-to-Use-PCR-Human-panel-I + II-V1.M). By qRT-PCR, these miRNAs were further assessed in training (30 GC VS. 30 NCs) and testing stages (71 GC VS. 61 NCs). We discovered a plasma miRNA signature including five up-regulated miRNAs ( Gastric cancer (GC) is the fifth most common malignancy and the third leading cause of cancer-related death all around the world in 2012. Approximately 50% of cases occur in Eastern Asia (the majority of which occur in China)456MiR-16 was used as a reference gene in some studies121415Recent research has demonstrated that circulating miRNAs that originate from tumors can be stably detected in peripheral blood and may aid in the detection and diagnosis of various types of cancer810In the current study, we performed plasma miRNA profiling through the quantitative reverse transcription polymerase chain reaction (qRT-PCR) based miRCURY platformA total of 242 subjects, including 133 GC patients and 109 normal controls (NCs), were enrolled in our study to assess the differently expressed miRNAs in the peripheral plasma of GC patients. As shown in Based on the qRT-PCR platform, the Exiqon miRCURY-Ready-to-Use-PCR-Human-panel-I + II-V1.M was utilized to analyze 168 miRNAs that are expressed in plasma/serum. This method was used to identify differently expressed miRNAs between 3 peripheral plasma pools from 30 GC cases and 1 pooled sample from 10 controls. Each miRNA was assayed twice on 384-well plates by qRT-PCR and the miRNAs with cycle threshold (Ct) value less than 37 and 5 lower than negative control  in the panel were included in data analysis. Among the 168 miRNAs analyzed, the expression of 33 miRNAs  was altered with at least a 1.5-fold change in all 3 pooled GC samples compared to the NC pool sample. These miRNAs were chosen to further validation in the experiments outlined below.miR-185, miR-20a, miR-210, miR-25 and miR-92b) were consistent with those in the training stage  curves. The areas under the curve (AUC) were 0.65 : 0.57–0.72), 0.67 (95% CI: 0.61–0.74), 0.75 (95% CI: 0.68–0.82), 0.65 (95% CI: 0.58–0.73) and 0.69 (95% CI: 0.62–0.76) for miR-185, miR-20a, miR-210, miR-25 and miR-92b, respectively . The risk score function (RSF) for each subject was calculated and used to explore the sensitivity and specificity of the five-miRNA signature. The signature showed a greater ability than any individual miRNA in detecting GC in the combined cohorts with AUC of 0.77 . As shown in The association of the five peripheral plasma miRNAs with clinical stage was also evaluated for all 133 GC patients. However, none of the five miRNAs showed significantly different expression in patients with stage III + IV compared to those with stage I + II. Furthermore, we also compared the levels of the five miRNAs in plasma from GC patients with metastasis to those without. Similarly, no significant difference of the five miRNAs was observed between the two cohorts (data not shown).miR-185, miR-20a, miR-210, miR-25 and miR-92b was significantly higher in tumor than in normal tissues, and this correlation was consistent with results obtained from peripheral plasma. Fluorescence in situ hybridization (FISH) was conducted in an area containing at least 85% tumor in order to appropriately visualize the expression of miR-185, miR-20a, miR-210, miR-25 and miR-92b in FFPE tissues of GC patients . When compared the expression levels of the five miRNAs in arterial plasma from patients with different stages , all five miRNAs were found to be elevated but only miR-185 was significantly up-regulated in patients with stage III + IV compared to those with stage I + II  from GC patients demonstrated a significantly different expression level in peripheral plasma exosomes as compared to controls (Supplementary Table S6 online).To investigate the potential form of the identified miRNAs in peripheral plasma of GC patients, exosomal miRNAs were examined in ten peripheral plasma samples from GC patients and compared to ten NC samples. None of the five miRNAs . Among the five miRNAs, et al.miR-16 as a potential diagnostic marker for GC. However, miR-16 was usually used as a reference molecule in the circulation and could also be secreted from and influenced by haemolysis1220miR-185, miR-20a, miR-210, miR-25 and miR-92b were found to be significantly up-regulated in GC. The five-miRNA signature could accurately discriminate patients from normal individuals. However, some promising miRNAs demonstrated in previous studies22miR-21) was identified in the screening phase but was not applicable in the following stages of the experiment. The external validation stage further demonstrated the reliability of the diagnostic value of the five-miRNA signature. Of note, consistent miRNA expression in GC tissue samples suggested the important roles for these miRNAs in tumorigenesis and progression of the disease.In this study, we identified five miRNAs which were up-regulated in the peripheral plasma of GC patients compared to controls. The diagnostic role of circulating miRNAs in GC has been reported in some previous studies. In addition to population and sample diversity, differences in analytic methods may have also contributed to inconsistent results between studies. Compared to TaqMan platform that was used in the study of Zhu miR-185 seemed to play a controversial role in the development and progression of GC, suggesting its conflicting function in GC24miR-185, circulating miR-20a was reported to be a noninvasive biomarker of GC, and this finding was consistently demonstrated in three recent studies826miR-20a could promote growth, migration and invasion of GC cells and enhance the chemoresistance of GC cells to cisplatin and docetaxel28miR-17-92 cluster, miR-20a could also sustain the self-renewal function of GC stem cells and promote the proliferation of GC cells by targeting E2F transcription factor 1 (E2F1)miR-20a might serve as an oncogene in GC. MiR-210, as a typical hypoxia-related miRNA, was reportedly induced by a hypoxia inducible transcription factor (HIF) dependent mechanism31miR-210 was reported to be one of four serum miRNAs  which could serve as biomarkers for the early detection of diffuse-type GCmiR-210 was identified as a member of the pan-cancer oncogenic miRNA superfamilymiR-210 in GC has not been explicitly reported, but it may promote growth and metastasis in various other cancer types, serving as a negative prognostic factormiR-25 and miR-92b, Zhu et al.miR-25 might be a potential biomarker in the detection of GC through relative quantification. Additionally, Omura et al.miR-92b level could be used to predict relapse of GC after S-1 adjuvant chemotherapy. In our study, the five-miRNA signature was closely associated with a diagnosis of GC. Further research of these miRNAs in GC formation and development is warranted.Among the five up-regulated miRNAs, miR-20a could be used to facilitate the diagnosis of nasopharyngeal carcinomamiR-210 was found in pancreatic ductal adenocarcinomamiR-25 was identified as a diagnostic and monitoring biomarker in esophageal squamous cell carcinomaThe diagnostic value of the circulating miRNAs identified in our study was also evaluated in some other cancers. Up-regulation of circulating miR-20a, miR-210, and miR-92b were significantly up-regulated in the arterial circulation which might suggest that circulating miR-20a, miR-210, and miR-92b could be absorbed and transmitted to distant body sites and participate in biological processes . The relatively stable concentration of miR-25 (shown in Supplementary Table S5 online) in the arterial and peripheral venous plasma could reflect a relative balance of absorption and secretion of this particular miRNA in the circulation. On the other hand, miR-185 demonstrated a significant reduction in arterial plasma than that in peripheral venous plasma. And arterial miR-185 was the only miRNA that was found to be significantly associated with TNM stage. The phenomenon was interesting, fascinating and difficult to explain. We assumed that circulating miR-185 released by tumor might stimulate the further secretion of miR-185 according to some mechanism. However, the exact mechanism is needed to be further studied in the future. We further explored exosomal miRNAs from peripheral venous plasma to identify the potential form of the five miRNAs in circulation. However, no difference of them in peripheral plasma exosomes between GC patients and NCs was found. A representative study to characterize the extracellular form of circulating miRNA complexes in human plasma and serum recently revealed that the majority of miRNAs were co-purified with the Ago2 ribonucleoprotein complex but a minority of specific miRNAs (such as miR-1979 and miR-940) associated predominantly with vesicles like exosomes. Their results showed that the four miRNAs in our study  were co-purified with the Ago2 ribonucleoprotein complex other than exosomes in plasma while the form of miR-92b was undefined in the circulation. Our findings indirectly validated their results.Circulating miRNAs were believed to be passively leaked or actively transported from cells during tumorigenesis and packaged into small membranous vesicles or protected by the formation of a protein-miRNA complex4344In conclusion, we identified a five-miRNA signature in the plasma of GC patients which could serve as a non-invasive biomarker in the detection of GC, indicating its potential application of plasma miRNAs in the screening of GC patients to improve clinical outcomes.miR-191-5p, miR-423-5p, miR-425-5p and miR-93-5p. And normalized Ct (ΔCt) = average Ct (assay) – average Ct . The relative expression between GC patients and NCs was assessed by 2−ΔΔCt method.A total of 133 histopathologically confirmed GC patients and 109 healthy donors were recruited at First Affiliated Hospital of Nanjing Medical University between 2011 and 2013. In the initial screening phase , 30 periIn the training stage, 30 GC samples and 30 NCs were used to confirm the miRNAs discovered through screening phase by qRT-PCR. In the testing phase, the miRNAs identified in the training stage were further validated in plasma samples of 71 GC patients and 61 NCs. An external cohort including 32 cases and 18 controls were analyzed to assess the diagnostic value of the five-miRNA signature in GC. An additional of thirty pairs of GC tissue specimens and matched normal tissues were obtained from surgery patients and used to verify the expression level of identified miRNAs. Arterial blood samples from another 38 GC patients were collected to compare the difference of miRNAs between peripheral and arterial plasma. And exosomal miRNAs from ten patients and NCs were further assessed to explore the potential form of the miRNAs in the peripheral plasma.Blood samples of healthy controls and GC patients before initial treatment were collected by using ethylenediaminetetraacetic acid (EDTA)-containing tubes. Cell-free plasma was isolated from blood samples within 12 hours after collection using a two-step protocol  to prevent contamination by cellular nucleic acids and then stored at −80 °C until further processing. Tissue specimens were obtained from surgery patients and kept in liquid nitrogen.The study was approved by Institutional Review Boards of the First Affiliated Hospital of Nanjing Medical University and informed consent was taken from each participant. And the study was carried out in accordance with the approved guidelines by the Hospital Ethics Committee.Exosomes were extracted from peripheral plasma using ExoQuick Exosome Precipitation Solution  according to the manufacturer’s instructions. Precipitated from 100 μl ExoQuick exosome precipitation solutions and 400 μl plasma, exosome pellets were lysed in 200 μl RNase-free water for further analysis.miR-39  was added to each sample after the addition of denaturing solution . For tissue samples, total RNA was isolated using Trizol  according to the manufacturer’s instructions. Total RNA was eluted in 100 μl of RNase-free water and stored at −80 °C until analysis. The concentration and purity of the total RNA was evaluated by using the ultraviolet spectrophotometer. For plasma samples, total RNA with concentration <10 ng/ul was not included in the analysis. And the mean and standard deviation of concentration of plasma RNA was 31.23 ± 12.36 ng/ul (range: 15.73~48.62 ng/ul). Thus, no carrier molecule was added during RNA isolation.Total RNA from 200 μl plasma or exosomes was extracted using the mirVana PARIS Kit  according to the protocol. For normalization of the sample-to-sample variation, 5 μl of synthetic C.elegans The specific primers of reverse transcription (RT) and polymerase chain reaction (PCR) from Bulge-Loop™ miRNA qRT-PCR Primer Set  were applied to amplify miRNAs. The amount of PCR product was detected by evaluating the level of fluorescence emitted by SYBR Green . RT and PCR were performed as previously described18−ΔΔCt method relative to RNU6B (U6) and cel-miR-39The amount of miRNA in plasma samples was calculated based on a standard curve constructed with the use of synthetic miRNAs Antisense LNA-modified oligodeoxynucleotide probes were purchased from RiboBio . Ten tissue specimens of GC and matched normal tissues were fixed in 10% neutral buffered formalin for 24 hours and passed through dehydration, clearing, and paraffin-embedding steps. Approximately 5 μm thick sections from tissue blocks were de-paraffinized, washed, air-dried, and subjected to antigen retrieval in 0.1 mmol/L of citric acid. Subsequently, slides were rinsed, washed and subjected to a pre-hybridization procedure  in 4 × saline sodium citrate (SSC), 25 °C for 20 min) to block nonspecific binding of probes and minimize background signals. Hybridizations were then performed in hybridization buffer  in dark at 25 °C for 60 min. Slides were then washed in 2 × SSC with 0.5% Tween-20 three times for 5 min respectively at room temperature and immersed in phosphate buffered saline (PBS) solution followed by nuclear staining with 4’,6-diamidino-2-phenylindole (DAPI). Hybridizations were observed with WIB filters under fluorescence microscope Olympus AX-70.2 test. A formula for calculating risk score of miRNA for GC was developedS) of each miRNA was set as 1 if the expression level of the miRNA was greater than the optimal cutoff value of corresponding miRNA level in controls, otherwise as 0. The risk score function (RSF) for patient i was calculated by: Mann–Whitney test was used to compare the difference of miRNA concentration in different groups. Demographic and clinical characteristics among groups and their relationship with miRNA were compared using one-way ANOVA or χij) of miRNA j on patient i was weighted by Wj, the regression coefficient estimated by univariate logistic regression models for each miRNA. The receiver operating characteristic (ROC) curves were used to estimate the diagnostic value of the identified miRNAs on GC.Here, the score . A P-value <0.05 was considered to indicate a statistically significant difference.How to cite this article: Zhou, X. et al. Diagnostic value of a plasma microRNA signature in gastric cancer: a microRNA expression analysis. Sci. Rep. 5, 11251; doi: 10.1038/srep11251 (2015).


LED inter-lighting was provided from 4:00 am to 4:00 pm for the daytime treatments and from 10:00 pm to 10:00 am for the nighttime treatments. Plants exposed only to solar light were used as controls. Daytime LED inter-lighting increased the photosynthetic capacity of middle and lower canopy leaves, which significantly increased yield by 27% in winter; however, photosynthetic capacity and yield were not significantly increased during summer. Nighttime LED inter-lighting increased photosynthetic capacity in both winter and summer, and yield increased by 24% in winter and 12% in summer. In addition, nighttime LED inter-lighting in winter significantly increased the total soluble solids and ascorbic acid content of the tomato fruits, by 20 and 25%, respectively. Use of nighttime LED inter-lighting was also more cost-effective than daytime inter-lighting. Thus, nighttime LED inter-lighting can effectively improve tomato plant growth and yield with lower energy cost compared with daytime both in summer and winter.Greenhouses with sophisticated environmental control systems, or so-called plant factories with solar light, enable growers to achieve high yields of produce with desirable qualities. In a greenhouse crop with high planting density, low photosynthetic photon flux density (PPFD) at the lower leaves tends to limit plant growth, especially in the winter when the solar altitude and PPFD at the canopy are low and day length is shorter than in summer. Therefore, providing supplemental lighting to the lower canopy can increase year-round productivity. However, supplemental lighting can be expensive. In some places, the cost of electricity is lower at night, but the effect of using supplemental light at night has not yet been examined. In this study, we examined the effects of supplemental LED inter-lighting (LED inter-lighting hereafter) during the daytime or nighttime on photosynthesis, growth, and yield of single-truss tomato plants both in winter and summer. We used LED inter-lighting modules with combined red and blue light to illuminate lower leaves right after the first anthesis. The PPFD of this light was 165 μmol m The former is a thermally insulated and nearly airtight warehouse-like structure in which the environment for plant growth can be controlled as precisely as desired and used for commercial production of leafy greens , herbs  and transplants  . The latHowever, light is a limiting environmental factor in the winter  and it aHigh planting density reduces light distribution along the plant profile, which is associated with mutual shading . The undInter-lighting can be more effective than traditional top-mounted supplemental lighting  and imprLEDs are considered a suitable light source for inter-lighting  because In addition, energy efficient use of lighting can be achieved by adjusting the LED inter-lighting schedule so that more is used at night, because the price per unit kilowatt can be lower with off-peak time-of-use (TOU), which is mostly at night. Other industries have rescheduled their operations to take advantage of discounted off-peak rates to reduce their electricity bills . In greeSolanum lycopersicum L., cv. Sanbi) seeds were sown in 128-cell plug trays filled with vermiculite. After 2 days in a dark room at a temperature of 26°C for germination, the trays were transferred to a walk-in type environment-controlled growth chamber . Seedlings were cultivated using Otsuka nutrient solution with 2.0 ± 0.2 EC and 5.5 ∼ 6.0 pH supplied only once a day using ebb and flow system. In the growth chamber, temperature was 20/16°C day/night during the winter and 22/18°C day/night temperature in the summer, under cool white fluorescent lamps with 360 μmol m-2 s-1 PPFD and 1200 ppm CO2 concentration.Tomato . The greenhouse was equipped with heat pumps to keep the day/night temperature of 22/17°C in winter, and pad and fan system were used to keep the day/night temperature at 26/20°C during the summer. The irrigation system was a modified nutrient film technique (NFT) designed to distribute nutrient solution evenly to each plant through longitudinal flow of the nutrient solution, rather than flowing from the start to the end of the growing bed . Otsuka nutrient solution  was used for fertigation with an application schedule of 10 min with a subsequent 30 min break during the day and an hour break during the night. The pH was maintained at 6.0 at all times. Electrical conductivity was set at 2.5 dS mof fruit . To achiescribed .Supplementary Figure S1) with a PPFD of 165 μmol m-2 s-1 measured at 10 cm from the LED module. LED modules were positioned on both sides of the aisle at a distance of 50 cm from the stem , and at a height of 40 cm from the Styrofoam panel under which the root system was established .LED inter-lighting  was used to illuminate the understory leaves. The lamp spectrum was red and blue combined .Internode length and stem diameter under the fruit truss, leaf chlorophyll content, leaf area, leaf mass per unit area (LMA), and shoot dry weight were measured. Chlorophyll content was determined using a spectrophotometer, as described previously . Leaf arLight distribution along the plant profile was measured at each canopy level  with a quantum sensor . The sensor was positioned such that the angle of inclination was the same as that of the representative canopy leaves. For LED inter-lighting measurement was made while LED inter-lighting was in use. Solar irradiance alone was used as a control and was measured similarly but in the absence of LED inter-lighting.Photosynthetic rate was measured with a portable gas exchange system  as described previously , 2010. WDiurnal photosynthetic rate and PPFD were also measured under growth conditions using three LI-6400 gas exchange systems at the same time, one each for the control plants and plants treated with daytime and nighttime LED inter-lighting. Measurements were made using representative leaves from mid-canopy. Although the leaves were originally positioned at different distances from the LED module, the leaf cuvettes were set at a representative distance of 10 cm from the LED module. Leaves were inserted into the leaf cuvettes positioned at their original inclination angle. Diurnal PPFD was recorded by positioning quantum sensors next to each leaf subjected to diurnal photosynthetic rate measurement.The fresh weight of each fruit was recorded, and two quality parameters were measured as described in Electric energy consumption of the LED modules was measured with a multimeter and a clamp ammeter  as described previously in -1) = [increase in yield with LED treatment (kg m-2)]/[electric energy consumption (kWh m-2)].Electric energy use efficiency (kg kWh-1) = Electric use efficiency (kg kWh-1)/the conversion coefficient from electrical energy to photosynthetically active radiation energy  × increase in yield (kg m-2)]/[electricity used for LED lighting per crop season (kWh m-2) × price of electricity (Yen kWh-1) + LED depreciation cost per crop season (Yen m-2)].Cost performance (return/cost) = [price of tomato (Yen kgt-test (for two means).Data were examined using the statistical software SPSS version 21.0 ; the significance of mean differences was analyzed either with Tukey’s HSD test (for more than two means) or with Figure 1). Estimated diurnal changes in PPFD at three different canopy levels  indicated that only 33 and 18% of the daily light reached the middle and lower foliar canopy, respectively, compared with the total incident light at the top of the foliar canopy. Introducing LED inter-lighting significantly improved the light distribution to the middle and lower canopy leaves . The light distribution within mid-canopy leaves was twice as high in plants that had direct irradiance from LED inter-lighting than in controls that were only exposed to solar light.Without LED inter-lighting, light distribution along middle and lower canopy leaves were highly deteriorated (Supplementary Table S1). Similarly, the nighttime LED inter-lighting significantly increased average nighttime temperatures, by 7.1 and 6.8% in summer and winter, respectively. In contrast, the relative humidity was significantly higher in the controls than in plants receiving nighttime LED inter-lighting treatments, in both seasons (Supplementary Table S1).LED inter-lighting also altered micro-environments around the mid-canopy where there was direct irradiance. Daytime LED inter-lighting significantly increased average daytime temperature by 5.3 and 6.7% in summer and winter, respectively . The photosynthetic rates in response to growth condition PPFD were measured in mid-canopy leaves from control and both daytime and nighttime LED inter-lighting plants (Table 1). The mid-canopy leaf photosynthetic rate under plant growth conditions for controls at 200 μmol m-2 s-1 PPFD (without LED inter-lighting) was 4.3, and the rate for LED inter-lighting at 400 μmol m-2 s-1 PPFD was 5.2 for daytime inter-lighting and 6.4 μmol m-2 s-1 for nighttime inter-lighting (Table 1). The photosynthetic rate was higher in plants exposed to daytime LED inter-lighting than in controls. However, leaves treated with daytime LED inter-lighting had lower leaf photosynthetic capacity than control leaves , which offset the net photosynthetic rate under growth conditions (Table 1).The overall change in plant micro-environment due to nighttime LED inter-lighting was accompanied by a higher photosynthetic capacity of middle and lower canopy leaves in both summer and winter. In contrast, daytime LED inter-lighting had a positive effect on leaf photosynthetic capacity in the winter but not in the summer  increased the diurnal photosynthetic rate in mid-canopy leaves by 5 μmol m-2 s-1 on average, as compared with a rate of about -0.4 μmol m-2 s-1 in control plants . When both daytime and nighttime LED inter-lighting were turned off, between 4:00 pm and 10:00 pm, the leaf respiration rate was higher for leaves from plants exposed to daytime or nighttime inter-lighting than for control leaves . In addition, LED inter-lighting significantly increased chlorophyll content in both middle and lower canopy leaves, but not in top canopy leaves, , to which LED inter-lighting did not contribute PPFD . LED inter-lighting had no significant effect on leaf area index (LAI) (Table 2), but a significant increase in stem diameter and LMA with short internode length under the fruit truss was observed (Table 2).The increased light distribution along mid-canopy leaves due to daytime or nighttime LED inter-lighting . Fruit quality also significantly improved as a result of LED inter-lighting. Daytime LED inter-lighting increased total soluble solids by 8.2% in summer and 24% in winter. Nighttime LED inter-lighting significantly increased total soluble solids by 20% in winter, but the increase in summer was not significant . Winter LED inter-lighting significantly increased ascorbic acid content, by 24% for daytime inter-lighting and 25% for nighttime inter-lighting. However, neither treatment altered the ascorbic acid content during the summer .Most importantly, we found that daytime LED inter-lighting increased yield by 27% in winter, but had no effect in summer. However, nighttime LED inter-lighting increased yield in both winter and summer by 24 and 12%, respectively  and nighttime (0.8 kg m-2) LED inter-lighting (Supplementary Table S2). In summer, nighttime LED inter-lighting increased the yield by 0.3 kg m-2, but daytime LED inter-lighting was deleterious, decreasing yield by 0.3 kg m-2 (Supplementary Table S2). Light use efficiency during winter for both daytime and nighttime LED lighting was 8.6 and 7.7 g MJ-1, respectively. In summer nighttime LED lighting had 3.9 g MJ-1 light use efficiency, but daytime LED light had negative effect. The cost-performance analysis for LED inter-lighting shows that only winter nighttime LED lighting can effectively improve tomato yield with high cost performance , whereas summer nighttime and winter daytime LED lightings improved yield but the economic returns from the lighting were smaller than the running cost of electricity.Because we used LED inter-lighting for the same duration (12 h) for the daytime and nighttime treatments, the total electric energy used was similar, 72 kWh mDevelopment of plant factories in Japan brought single-truss tomato cultivation method for more improvement as an alternative system for its merits over multi-truss cultivation. However, at high planting density with a LAI higher than 3, light becomes a limiting factor for plant growth, because the leaf architecture and mutual shading prevent the light from penetrating to the lower canopy and thereby suppress photosynthetic activity . High plFigure 1), leading to a significantly higher photosynthetic rate compared to control leaves that were grown under only solar light . This was also supported by the data that LED inter-lighting enhanced the diurnal photosynthetic rate due to better vertical light distribution . Most importantly, nighttime LED inter-lighting significantly increased tomato yield during both winter and summer, although daytime LED inter-lighting increased tomato yield only in winter . Supplemental light is expected to increase leaf photosynthesis (Table 1) and led to no improvement in yield . Other studies has been reported that inter-lighting did not increase yield significantly (-2 s-1 in a greenhouse (-2 s-1 and the temperature exceeded 33°C during the middle of the day in the summer (Supplementary Table S1). In addition, daytime LED inter-lighting increased light distribution and temperature significantly around mid-canopy . Therefore, it is possible that the high temperature and high solar irradiation during mid-day in summer exceeded the optimal range for tomato production and thus reduced yield.Application of supplemental lighting when there is low light interception could improve canopy light interception, leaf chlorophyll content, and leaf photosynthetic capacity, as well as the assimilate supply to the fruit, resulting in enhanced crop yield . Our stuynthesis  and thusficantly . Trouwboficantly . It has ficantly . Usuallyeenhouse . In our Figure 6). Among environmental factors light intensity and temperature are the most important in determining the final ascorbic acid content . Nighttime LED inter-lighting had also higher light use efficiency. Previous study reported light use efficiency of tomato cultivation between 2.8 and 4.0 g MJ-1 . This suggests that whether or not to invest on supplemental LED lighting critically depends on reliable yield expectations and cost of LED for a specific condition. Fortunately, historical and projected evolution of LED is rapid and cost is decreasing; each decade, LED prices have fallen by a factor of 10 while performance has grown by a factor of 20 (Energy is second only to labor as the highest expense in greenhouse production , and sup0 g MJ-1 . In our or of 20 .Daytime LED inter-lighting to enhance light distribution of the lower canopy leaves did not lead to higher photosynthetic capacity and yield in summer. However, photosynthesis, growth, and yield of single-truss tomatoes were improved when LED inter-lighting was provided during the daytime in winter. Nighttime LED inter-lighting had a positive effect on photosynthesis, growth, and yield in both seasons. Our results suggest that nighttime LED inter-lighting can be used cost efficiently to increase yields by taking advantage of off-peak electricity pricing, both in summer and winter. This is the first report that provides an evidence on LED inter-lighting at nighttime to take advantage of off-peak electricity discount rate.FT, NL, and WY conceived and designed the experiments. FT performed the experiments. FT analyzed the data. FT, NL, and WY prepared the manuscript, and FT, NL, KS, TM, MT, TK, and WY contributed extensively to its finalization.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objectives: The aim was to assess the reliability and validity of the quality of life (QoL) instrument CASP-19, and three shorter versions of CASP-12 in large population sample of older adults from the HAPIEE  study.Methods: From the Czech Republic, Russia, and Poland, 13,210 HAPIEE participants aged 50 or older completed the retirement questionnaire including CASP-19 at baseline. Three shorter 12-item versions were also derived from original 19-item instrument. Psychometric validation used confirmatory factor analysis, Cronbach's alpha, Pearson's correlation, and construct validity.Results: The second-order four-factor model of CASP-19 did not provide a good fit to the data. Two-factor CASP-12v.3 including residual covariances for negative items to account for the method effect of negative items had the best fit to the data in all countries . Goodness-of-fit indices for the two-factor structure were substantially better than second-order models.Conclusions: This large population-based study is the first validation study of CASP scale in Central and Eastern Europe (CEE), which includes a general population sample in Russia, Poland, and the Czech Republic. The results of this study have demonstrated that the CASP-12v.3 is a valid and reliable tool for assessing QoL among adults aged 50 years or older. This version of CASP is recommended for use in future studies investigating QoL in the CEE populations. With declining mortality rates and increased longevity, there has been a substantial increase in the proportion of adults reaching older age. In Europe, the number of older people aged 65 and over has risen significantly over the past two decades. The percentage of Europeans aged 65 or older is projected to rise from 17.4% at present to 27% in 2058 . CASP-19 is a theoretically based measure of broader QoL, which was specially designed to measure QoL in the ‘Third Age’  waves 1–5 . In addition, factor structure of the newly suggested CASP-12v.3 instrument will be further investigated using CFA.In the past 20 years, the countries of CEE have experienced a remarkable social and economic transition from closed, totalitarian, and centrally planned economies towards open, democratic, and market-based economies. Such transition has had devastating impact on health. In the early 1990s, many countries in CEE experienced a dramatic increase in mortality rates. The largest increase was concentrated in the Former Soviet Union (FSU). In Russia, for example, male life expectancy at birth decreased from 63.8 to 57.7 years and 74.4 to 71.2 years for women between 1990 and 1994  project. The sampling procedure and design of HAPIEE is described in detail elsewhere , divorced, and widowed), the educational level was categorised into four groups , health status was divided into three groups , level of material deprivation was assessed by three questions about the frequency of difficulties in (1) paying bills, (2) buying food, and (3) clothes necessary for themselves and/or his family. The answers were recorded on a 5-point scale (coded from 0 to 4); the total deprivation score was calculated as the sum of the three questions, and categorised into three groups: low (0), medium (1–6), and high (7–12). Self-reported economic activity was classified into the following categories: working pensioners and non-working pensioners.Participants in Poland and the Czech Republic completed the questionnaire during a nurse visit to their home (85% of them subsequently attended an examination in a clinic); all Russian participants completed the questionnaire during a visit to a clinic.Data were analysed in STATA version 12 for descriptive analyses and Mplus version 6.11 for CFAs . It evaluates the extent to which items within a scale are inter-correlated with one another and measures the same concept. Cronbach's alpha typically ranges from 0 to 1. Internal consistency reliability is suggested to be acceptable when Cronbach's alpha ≥ 0.70  single-factor model, (2) first-order model, and (3) second-order model. A schematic representation of the models can found in A single-factor model where all 19 or 12 items load directly onto unobserved variable called QoL was tested , followeCFA was computed using the weighted least square estimator with a mean- and variance-adjusted chi-squared method to handle ordered categorical items as dependent variables in Mplus. Missing data across CASP-19 were handled using full information maximum likelihood estimation. This method computes parameter estimates on the basis of all available data, including the incomplete cases. The procedure works under the assumption that the data are missing at random.To evaluate overall model fit, three goodness-of-fit indices were calculated. These indices include comparative fit index (CFI), Tucker–Lewis index (TLI), root mean square error of approximation (RMSEA), and weighted root mean square residual (WRMR). According to Hu and Bentler , a CFI vThe study received ethical approval from the local ethical committees in each participating country and at University College London, and all participants gave written consent.n = 3782; Russia: n = 3802; Poland: n = 5626).Of all the 14,059 retirees , we restricted inclusion in the study to those aged 50–70 years who answered at least one of the CASP-19 items. There were 449 retirees who did not give any responses to the CASP-19 questionnaire and were, therefore, excluded from the analyses. Also, there were participants younger than 50, who answered the module for retired people. These respondents are most likely to be retired for health reasons. There were also few respondents aged 70 or older but their number was low and they would not well represent this age group. Consequently, 400 subjects who were outside the 50–70 years range were excluded. Thus, the analytical sample consisted of 13,210 individuals . Also, the Russians reported higher levels of self-reported material deprivation. The proportion of participants with university education was higher in Russia, and in all countries it was higher in men than in women. Compared to Czech and Polish women, there was a large proportion of widows and lower proportion of married or cohabiting women in Russia. Four times as many Russian women (29.3%) were widowed, compared to men (7.3%). In regards to economic status, the Russian sample consisted only of non-working pensioners, while proportion of Czech and Poles still working ranged between 7.7% and 13.5%, respectively.There were differences in socio-demographic characteristics between the samples. Notably, Russians reported higher levels of poor/very poor health than Czechs (women 35.0% vs. 14.2%), while also presenting lower rates of very good/good health . Men scored significantly higher on the CASP-19 than women in all countries. Polish men and women reported the highest CASP-19 scores  for men and 36.8  for women). The lowest CASP-19 scores were reported by Russian men ). Distribution of CASP-19 was skewed in all countries . In the Czech sample, the median CASP-19 score was 36. Median scores for each CASP-19 sub-scale were as follows: control = 6 (inter-quartile range [IQR] = 4–8), autonomy = 9 (IQR = 7–11), self-realisation = 8 (IQR = 6–10), and pleasure = 13 (IQR = 11–15). Among Russian participants, median scores for each CASP-19 sub-scale were as follows: control = 6 (IQR = 4–8), autonomy = 8 (IQR = 7–10), self-realisation = 7 (IQR = 4–10), and pleasure = 13 (IQR = 10–15). In Poland, median scores were control = 7 (IQR = 5–9), autonomy = 8 (IQR = 6–10), self-realisation = 10 (IQR = 8–12), and pleasure = 14 (IQR = 12–15).There were significant differences in mean values of CASP between countries . Missing data were relatively small, with between 0.5% and 6.7% respondents not providing a response to an item. A marked ceiling effect was found in the pleasure domain, with the highest ceiling effect of 67.3% (Czech Republic), 70.4% (Russia), and 76.2% (Poland).The distributions of responses to each of the items in the CASP are shown in Appendix 2. Most of the participants completed all 19 items .p < 0.001.The associations of CASP-19 dimensions with physical functioning scales, self-rated health, and CESD-20 are shown in The four-factor solutions for CASP-19 had relatively poor model fit, as illustrated by the goodness-of-fit indices. RMSEA values were all above or equal to 0.10; CFI and TLI values were below 0.90. Although the three-factor second-order model suggested the best fit of all the three models, the fit indices indicted only an acceptable model in the Czech and Polish samples  and a marginal model fit for Russia .In addition to the established measurement models, a model composed of control/autonomy and self-realisation/pleasure factors which included error correlations between negative items were tested. Two-factor model of CASP-12v.3 was good fit to the data in the Czech Republic  and satisfactory model fit in Poland and Russia . Goodness-of-fit indices for the two-factor structure were substantially better than the second-order models . Similarp < 0.001). Items on the self-realisation/pleasure exhibited strong factor loadings (>0.40) for all the three samples. Four items were below the 0.4 level in the Czech sample , whereas two items did not reach the recommended 0.4 threshold in the Russian and Polish samples  . This indicates that there may be only one factor underlying the 12 items of CASP.For two-factor measurement models of CASP-12v.3, all item-factor loadings were significant (ely) see . Item C1The purpose of this research was to evaluate the psychometric properties of the CASP scales in a sample of older adults aged 50 or over living in CEE. This large population-based study in CEE is the first validation study of CASP scales in this population group, which includes a general population sample in Russia, Poland, and the Czech Republic.Given the mortality crisis, higher levels of cardiovascular diseases, and the high prevalence of unhealthy behaviours in Russia  trends, socio-economic trajectories, and alcohol consumption patterns in the region. For example, in 2011, the life expectancies at age 45 years in Russia, Poland, the Czech Republic, and the European Union were 28.6, 33.1, 34.0, and 36.4, respectively (WHO, The study has a number of limitations. First, the CASP-19 is a self-completed questionnaire. A methodological problem commonly associated with the use of self-report measures, which may have been present in our study, is the inability to determine the extent to which responses accurately reflect the respondents’ experiences due to inaccurate recall; respondents for various reasons may under or overestimate their QoL. Second, since the Russian data only comprised non-working subjects, the working pensioners are excluded from the analysis. Consequently, respondents included in the study may not be representative of the whole population and the generalisation of our results may be limited. Thus, future studies with a more heterogeneous group of participants are needed to examine the psychometric properties in more detail. Finally, the data used in this study had been collected in 2002–2005, and the results reflect conditions in these countries at the time of data collection which might be little different from the conditions in these countries now. We, however, believe that 15 years between the start of political and social changes and data collection have been long enough to make these societies more stable and the results are still applicable to current societies in the region.Despite the above-mentioned limitations, this is one of the first, and the largest study so far on the levels and psychometric properties of CASP in CEE. In conclusion, CASP-12v.3 is a valid and reliable tool for assessing QoL among older adults aged 50 years or older. This version of CASP is recommended for the use in future studies investigating QoL in the CEE populations.